Protein—protein interactions are known to play an important role with respect to structure and functions of living cells.
Many problems currently studied in molecular biology and biochemistry, such as gene transcription mechanism and intracellular information signaling, are related to protein—protein interactions.
Some of the problems in the field of molecular biology and biochemistry have been heretofore solved by the development of a two-hybrid method (Chien, C. T., Bartel, P. L., Sternglanz, R., Fields, S., Proc. Natl. Acad. Sci. USA 1991, 88, 9578–9582; Fields, S., Song, O., Nature 1989, 340, 245–246) by which screening is conducted based on interaction of a “bait” protein and a “prey” protein in a protein library. The two-hybrid method has been suggested as an effective method that facilitates the identification of candidate molecules for protein—protein interaction and also creates new protein—protein interaction maps (Flores, A., Briand, J. F., Gadal, O., Andrau, J. C., Rubbi, L., Mullem, V., Boschiero, C., Goussot, M., Marck, C., Carles, C., Thuriaus, P., Sentenac, A., Werner, M., Proc. Natl. Acad. Sci. USA 1999, 96, 7815–7820; Ito, T., Tashiro, K., Muta, S., Ozawa, R., Chiba, T., Nishizawa, M., Yamamoto, K., Kuhara, S., Sakaki, Y., Proc. Natl. Acad. Sci. USA 1999, 97, 1143–1147; Walhout, A. J. M., Sordella, R., Lu, X., Hartley, J. L., Temple, G. F., Brasch, M. A., Thierry-Mieg, N., Vidal, M., Science 2000, 287, 116–122). However, the problem associated with the two-hybrid method is that it is applicable only to the analyzable protein interactions that occur close to the reporter gene in cell nuclei and, therefore, this method lacks generality.
Reliability is yet another problem associated with the two-hybrid method, and a confirmation test using proteins with known functions has to be conducted by employing model cells or animals for every assay (Walhout, A. J. M., et al., Science 1999, 287, 116–122).
Accordingly, novel methods for protein—protein interaction analysis have been suggested, those methods including a ubiquitin split protein sensor (USPS) method by which N- and C-terminal ubiquitins through reactions with proteins and a nuclear-localized reporter is activated by cleavage of a transcription factor (Dunnwald, M., Varshavsky, A., Johnsson, N., Mol. Biol. Cell 1999, 10, 329–344; Johnsson, N., Varshavsky, A., Proc. Natl. Acad. Sci. USA 1998, 95, 5187–5192; Stagljar, I., Korostensky, C., Johnsson, N., Heesen, S., Proc. Natl. Acad. Sci. USA 1998, 95, 5187–5192; and an SOS recruit system, in which a catalyst domain is brought close to a membrane localization domain through protein—protein interaction, a guanine exchange factor (GEF) or Ras is reconstructed, and it further complements yeast temperature-sensitive mutated yeast GEF (Aronheim, A., Nucleic Acids Res. 1997, 25, 3373–3374; Aronheim, A., Zandi, E., Hennemann, H., Elledge, S. J., Karin, M., Mol. Cell. Biol. 1997, 17, 3094–3102; Broder, Y. C., Katz, S., Aronheim, A., Curr. Biol. 1998, 8, 1121–1124).
As a more general approach, a split enzyme technology method has been reported (Rossi, F., Charlton, C. A. and Blau, H. M., Proc. Natl. Acad. Sci. USA 1997, 94, 8405–8410; Remy, I., Michnick, S. W., Proc. Natl. Acad. Sci. USA 1999, 96, 5394–5399; Pelletier, J. N., Arndt, K. M., Pluckthun, A., Michnick, S. W., Nature Biotech. 1999, 17, 683–690). With this method, a split enzyme is reconstructed through protein—protein interaction and its enzymatic activity is restored. The activity of the reconstructed enzyme can be measured by fungi or cellular phenotype or by a fluorescent enzyme substrate analogues.
These methods have a comparatively high accuracy and attain the object of assaying interactions between intracellular proteins or membrane proteins, but they are applicable to only appropriately designed cells. Another problem is that sufficient accuracy and sensitivity cannot be obtained. Still another problem is that such methods require a variety of substrates to conduct the analysis and are therefore troublesome.
Therefore, a general method or probe suitable for accurate and simple analysis of protein—protein interactions and applicable to any protein has not yet been developed.
With the foregoing in view, it is an object of the invention of the present application to solve the problems of the prior art and to provide a probe for protein—protein interaction analysis, which enables highly accurate and simple analysis of protein—protein interactions, and to a method for the analysis of protein—protein interactions using such a probe.